The efficient population of the triplet excited states in heavy metal-free organic chromophores has been one of the long-standing research problems to molecular photochemists. The negligible spin–orbit coupling matrix elements in the purely organic chromophores and the large singlet–triplet energy gap (ΔE_S–T) pose a hurdle for ultrafast intersystem crossing (ISC). Herein we report the unprecedented population of triplet manifold in a series of nitrogen-annulated perylene bisimide chromophores (NPBI and Br-NPBI). NPBI is found to have a moderate fluorescence quantum yield (Φ_f = 68 ± 5%), whereas Br-NPBI showcased a low fluorescence quantum yield (Φ_f = 2.0 ± 0.6%) in toluene. The femtosecond transient absorption measurements of Br-NPBI revealed ultrafast ISC (k_ISC = 1.97 × 10¹⁰ s⁻¹) from the initially populated singlet excited state to the long-lived triplet excited states. The triplet quantum yields (Φ_T = 95.2 ± 4.6% for Br-NPBI, Φ_T = 18.7 ± 2.3% for NPBI) calculated from nanosecond transient absorption spectroscopy measurements showed the enhancement in triplet population upon bromine substitution. The quantum chemical calculations revealed the explicit role of nitrogen annulation in tuning the excited state energy levels to favor the ISC. The near degeneracy between the singlet and triplet excited states observed in NPBI and Br-NPBI (ΔE_S–T = −0.01 eV for NPBI, ΔE_S–T = 0.03 eV for Br-NPBI) facilitates the spin flipping in the molecules. Nitrogen annulation emerges as a design strategy to open up the ISC pathway and the rate of which can be further enhanced by the substitution of a heavier element.